This invention pertains to instruments of simple design and operation used to measure the thickness of thin films as a function of changes to or attenuation of incident light. The devices and methods of this invention are improvements of a fixed polarizer ellipsometer where film thickness can be related as a function of the degree of ellipticity in polarized light, or rotation of polarized light, that is reflected by a thin film. More specifically, the invention relates to improvements in ellipsometry that speed measurement acquisition time and reduce the cost of ellipsometry devices for use in specific binding assays and other applications. The invention also relates to instruments and methods where measurement of light attenuation by a thin film is no longer dependent on the generation of elliptically polarized light.
The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.
Optical measurements are commonly used to determine the thickness of thin films. Ellipsometers provide this information by determining the degree of ellipticity in polarized light that is reflected from a thin film. Ellipsometers typically include a light source, a polarizer, an analyzer, an optical compensator or quarter wave plate, and a detector. For example, U.S. Pat. No. 5,936,734 discloses using singly, partially, and/or multiply polarized electromagnetic radiation for ellipsometrically measuring regions of a patterened sample system, while U.S. Pat. No. 5,946,098 discloses a modified ellipsometer comprising a retarder element in the form of a prism.
Complex mathematical calculations are conventionally used to determine the film thickness. To use these mathematical calculations, the ellipsometer must have precise alignment of the rotating components for the measurement of signal intensity at the detector. Expensive and precise optical components must be used to provide for optimization of the detector signals in performing film thickness measurements. Measurement time is slow but the ellipsometer can provide precise thickness and refractive index determinations.
The rotation of ellipsometer components can be used to provide a sinusoidal plot of intensity at the detector as a function of time and angular velocity of the rotating component, as disclosed in U.S. Pat. No. 5,581,350. Measurements are made at two or more analyzer angles to determine the angle of the polarizer""s optical axis and the offset of the actual analyzer angle relative to its nominal angle. Measurements provide the angle of the analyzer""s optical axis and the offset of the polarizer angle relative to its nominal angle. This information is used to calibrate the ellipsometer, but the device operates according to time-consuming traditional principles that require precise alignment of rotating components for purposes of measuring film thicknesses. Similarly, U.S. Pat. No. 5,877,859 discloses rotating compensator ellipsometry methods, relying on a rotating compensator to produce a signal having a dc component, a two omega component, and a four omega component.
According to U.S. Pat. No. 3,985,447 it is also possible to rotate both the optical compensator and the polarizer at different angular speeds to measure the resultant transmitted optical intensity as a function of time. A Fourier analysis is used to determine the Stokes parameters of light that is reflected by the thin film. The film thickness and refractive index of the film can also be calculated in this manner based upon the Stokes parameters. A disadvantage of this device is that the system requires additional components including a time dependent rotating compensator. These additional components increase the expense and complexity of the system.
U.S. Pat. No. 4,725,145 discloses an instrument and method of use for measuring the state of polarization. The instrument contains only a photodetector. The photodetector has a partially specular surface and is placed at an oblique angle relative to the incident light source. The light adsorbed by the photodetector generates an electrical signal that is detected and related to the polarization of the light. The detector may be rotated to determine if the light contained any elliptical character. In the preferred mode of operation the entire system is rotated. The improvement is that the instrument does not include any wave retarders or polarizers. The system may contain one or more photodetectors. The amount of light adsorbed is a fraction of the incident radiation and is dependent on the incident light source and the azimuthal orientation of the plane of incidence. The detector surface rotates in a conical manner. Thus the plane of incidence is a plane which revolves around and through the incident light. The electrical output is modulated by the rotation and thus the modulation is a measure of the state of polarization of the light incident on the detector.
U.S. Pat. No. 5,552,889 discloses a method for measuring changes in polarized light that is independent of temperature. The method examines the AC and DC components of the light separately. The method requires an instrument design where two or more polarizers are arranged so that they are not orthogonal to each other. The modulation of the polarized signal is then measured at one or more photodetectors. The intensity of a constant component of the polarization signal is related to the position of the average plane of polarization. The alternating component of the polarization signal is normalized to the constant component and then the phase, amplitude, and position of the polarization is determined. The polarization signal is exactly linearized. The method requires a beam splitter to produce two beams of light.
U.S. Pat. No. 5,625,455 discloses an ellipsometer and an ellipsometric method. In the method the complex dielectric constant, the complex index of refraction, the transmittance, the reflectance, the adsorption coefficient, the optical density, and other optical properties may be measured by reflectance of a monochromatic light source. The instrument and method of use provide a direct measure of the optical and spectroscopic properties of the sample without numerical approximation or wavelength frequency scans. The light source should be elliptically polarized and the angle of incidence should be between 0xc2x0 and 90xc2x0. The digitized intensity data reflected or transmitted from the sample is analyzed using integrals or sums. The integrals eliminate noise and allow the method to start and stop at any analyzer angle.
In certain applications, it is possible to eliminate some of the components from an ellipsometer-like device for purposes of reducing the cost of manufacture while still providing an acceptable level of accuracy. U.S. Pat. No. 5,494,829 to Sandstrom et al. describes a device that operates according to the principles of ellipsometry, but has a fixed polarizer and a fixed analyzer with the additional cost advantage of having no optical compensator or other complex optical components. This device is used for binding assay analysis to determine whether a biochemical reaction has provided a thin film analyte indicating the presence of bacterial infection in a patient.
According to the ""829 patent, an antigen or an antibody is bound to a substrate and is incubated with an analyte solution that is prepared to include a body fluid specimen from a patient who is being tested for infection. A biochemical reaction grows a thin film on the substrate if the corresponding antibody or antigen is present in the solution. A positive test result is indicated by signal intensity at the detector relative to a delimiting threshold or background value. The device is typically calibrated to measure thickness in films having been produced by a particular antigenic reaction.
A number of spectrophotometric systems have been designed to analyze the thickness of films, in particular photoresist films. These instruments require complex arrangements of optical components or focus on specific geometric features in the film. The instruments may require detection of more than one wavelength or angle to determine the film thickness. Several of the methods will not work without exact information on the refractive index of the film. The spectrophotometers do not measure film thicknesses well on optical substrates with low reflectivity. The most significant limitation is the poor signal to noise ratio obtained with these instruments. These instruments also have difficulty measuring films such as amorphous silicon.
For example, U.S. Pat. No. 4,680,084 describes a very complicated instrument that uses multiple light sources and lens and beam splitters and more than one detector to determine the thickness of a film. In addition the method requires that the optical substrate have patterned features present that are opaque to the incident light. These features are used to correct the detected signal for contributions unrelated to the film thickness. U.S. Pat. No. 4,618,262 describes a laser-based interferometer that measures an etching process depth by the use of specific features on the optical substrate to determine when the etch process is complete. In this method the distance between adjacent maxima are used to determine the etching rate. The characteristic sinusoidal pattern terminates when the etch process reaches the optical substrate. This method must be able to resolve etch features on the order of 1-3 microns which is problematic because the laser beam is on the order of 700 microns in diameter. Thus resolution of these small features from background is difficult. Optical components have been added to the system to address this issue.
U.S. Pat. No. 5,494,829 describes the use of a simple calorimeter or reflectometer to measure a color change or a change in intensity for interpretation of binding assay results. The signal is a function of a change in wavelength or the change in intensity of a range of wavelengths where the optical substrate is designed to generate a visual interference effect and thus change color as a function of thickness change.
Each of the foregoing U.S. Patents describing the background of the invention is hereby incorporated by reference in its entirety, including all tables, figures, and claims.
There remains a need to provide inexpensive ellipsometric instruments that measure film thickness with acceptable accuracy for a variety of binding assays or other applications. This need may be addressed by ellipsometry, or other thin film mechanisms for light attenuation, where the device is capable of measurement without undue time being spent to align system components and without unnecessary optical components that reduce system efficiency. There also remains a need to provide inexpensive reflectometric instruments that measure film thickness with acceptable accuracy, without necessarily providing an absolute thickness determination, for a variety of binding assays and other applications according to the principles of multilayer film reflection theory. The reflectometric instruments should also be capable of performing measurements without undue time being spent to align system components, to acquire signals, or to analyze signals. The instruments of this invention are easy to operate and the data interpretation is straightforward. The instrument performance is highly predictable as the response to any particular binding assay system can be modeled and the instrumentation is tailored to analysis a particular binding assay surface construction or a range of similar constructions.
The invention provides devices and methods for use in measuring film thickness comprising an AC mode fixed polarizer ellipsometer. Use of the AC mode eliminates the need for the precise alignment of the polarizing elements, thus reducing the cost of manufacturing the device. In the AC mode, one of the polarization elements, e.g., the analyzer or the polarizer, is rotated in complete cycles at a constant speed in order to generate an alternating signal at the detector element. The device improves the signal over existing fixed angle polarizer ellipsometers because the change in signal versus the change in thickness produces a steeper slope (i.e., a larger signal difference for a change in thickness), and thus a better signal to noise ratio. As the analyzer or polarizer rotates, the signal received from a test surface varies with the rotation of the analyzer (polarizer), or varies as a function of time. The signal observed is thus a quasi-sinusoidal curve with an amplitude and phase that is characteristic of the film being analyzed. The data analysis can utilize any combination of features in the signal generated, but preferably uses a peak to peak difference as the output for a specific thin film. For instance, an average of all the peak signal strengths can be made and reported as the sample value. Exact thickness determinations can be made by comparison of the peak to peak value for an unknown sample relative to a standard curve of instrument output versus known film thicknesses. The standard curve is based on a film that is similar or identical in properties to the thin film to be analyzed and that is deposited on a substrate with the same structure as the substrate used with the test film. The standard curve can also be created by theoretical calculations. The AC mode ellipsometer is designed such that all instrument parameters are modeled to allow maximum thickness differentiation over a pre-determined range of thickness for a given optical support and thin film layer(s) combination.
Other instrument embodiments (reflectometric) involve the use of one polarizer or no polarizer, respectively, in the measurement of a change in the characteristics of light reflected from an optical substrate supporting one or more thin film. By eliminating one or both of the polarizers, the reflectometric instrument design is both less complex and less expensive. The removal of the polarizing elements also provides an advantageous increase in signal intensity, because any optical element will introduce some insertional loss of signal. The single polarizer instrument is preferably used when only one component (i.e., the s or p polarization component) of the incident light is to be used. In the single polarizer instrument either the polarizer or the analyzer may be removed.
Moreover, at a steep angle of incidence there is very little operational difference in the s- and p-components of light. Accordingly, if both components are used to measure film thickness, for example in a polarizer-free device, the instrument performs equivalently to a device comprising a polarization element. Thus, in other embodiments, the invention concerns polarizer-free devices which use unpolarized light at steep angles of incidence. The polarizer-free instrument is designed to analyze a specific optical substrate in order to measure a film created by the binding of a biological material, or a film created in other binding assays, over a range of film thicknesses. The thickness range to be accommodated depends on the type of binding assay to be performed. Based on empirical observation or theoretical calculations from thin film reflection theory, the proper wavelength of incident light and angle of incidence can be selected. Thus, a single instrument design can be selected that will accommodate a number of different binding assays based on a similar optical substrate.
In other preferred embodiments, the invention also provides methods of using any of the devices described herein to relate a change in light intensity to a change in film thickness. An exact thickness determination can be made by comparison to a standard intensity curve generated with known film thicknesses, where the standard curve is created with a known film that is optically similar to the test film. The detector signal intensity may be used without correction, or following correction using a comparative detector signal intensity (i.e., application of a normalizing function) obtained from a negative binding control sample, or other background measurement.
The present invention overcomes the problems known to those skilled in the art that are outlined above, and advances the art by providing cost-effective devices that measures film thickness with acceptable accuracy for a variety of binding assays and other applications according to the principles of ellipsometry. These advantages are obtained by rotating the analyzer or the polarizer to produce a quasi-sinusoidal intensity in reflected light from the sample under test and by mapping selected intensity values to a film thickness through the use of a standard curve or other reference data. This concept permits the devices described herein to perform film thickness measurements without undue time being spent to align system components and without the use of complex mathematics and optical components. The system is inexpensive to manufacture because it does not require the use of an optical compensator, quarter wave plate, or other precision optical components and component alignment.
Thus, in a first aspect, the invention describes devices for use in determining a film thickness of a sample. The devices can comprise a substrate for supporting the sample; a light source for producing electromagnetic radiation to illuminate the sample; a first polarization element located between the light source and the sample; a detector for detecting electromagnetic radiation reflected from the sample; and a second polarization element located between the detector and the sample. At least one of the first and second polarization elements can be rotated to vary an s and/or p content of the electromagnetic radiation with time. The signal obtained from the detector is used to determine film thickness by a method comprising the use of a standard function that correlates film thickness to detector signal intensity.
In particularly preferred embodiments, one or more of the following can be included in the devices: (i) a light source that produces monochromatic electromagnetic radiation, (ii) electromagnetic radiation selected from the group comprising visible light, infrared light, and ultraviolet light, (iii) a first polarization element comprising a rotatable polarizing filter, (iv) a second polarization element comprising a rotatable polarizing filter, (v) a first polarization element comprising a rotatable polarizing filter, and a second polarization element comprising a fixed analyzer, (vi) a first polarization element comprising a fixed polarizing filter, and a second polarization element comprising a rotatable analyzer, (vii) rotating at least one of the first and second polarization elements to provide a quasi-sinusoidal intensity signal at said detector, (viii) relating a film thickness to an amplitude of the quasi-sinusoidal intensity signal, (ix) relating a film thickness to a peak to peak amplitude of the quasi-sinusoidal intensity signal, (x) a control sample comprising a known film thickness, (xi) a control sample that is a negative control sample, (xii) a standard function comprising a normalizing function which relates the detector signal intensity to a comparative detector signal intensity obtained from the negative control sample, and (xiii) a normalizing function that is a ratio of the detector signal intensity and a comparative detector signal intensity obtained from the negative control sample.
The term xe2x80x9csamplexe2x80x9d as used herein refers to any material which can be deposited on the surface of a substrate to form a film. Preferred samples can be organic materials such as biological materials (e.g., nucleic acids, antibodies, antigens, receptors, analytes, chelators, enzyme substrates, etc.), or inorganic materials such as silicon oxide, silicon dioxide, silicon nitride, etc. A sample preferably can be a solution containing such materials. The term xe2x80x9cnegative control samplexe2x80x9d as used herein refers to any substrate which lacks a thin film. Such a negative control sample can be used to provide a baseline or comparative signal from the device.
The terms xe2x80x9cfilmxe2x80x9d and xe2x80x9cthin filmxe2x80x9d as used herein refer to a one or more layers of sample material deposited on a substrate surface. A film can be about 1 xc3x85 in thickness, about 5 xc3x85 in thickness, about 10 xc3x85 in thickness, about 25 xc3x85 in thickness, about 50 xc3x85 in thickness, about 100 xc3x85 in thickness, about 200 xc3x85 in thickness, about 350 xc3x85 in thickness, about 500 xc3x85 in thickness, about 750 xc3x85 in thickness, about 1000 xc3x85 in thickness, and about 2000 xc3x85 in thickness. Particularly preferred are films from about 5 xc3x85 to about 1000 xc3x85; most preferred are films from about 5 xc3x85 to about 350 xc3x85.
The terms xe2x80x9csubstrate,xe2x80x9d xe2x80x9coptical supportxe2x80x9d and xe2x80x9csupportxe2x80x9d as used herein refer to a support within a device for a sample under study. Suitable substrates can be made of any reflective material known by those skilled in the art, and provide a planar surface upon which a sample film is deposited. Preferably, the substrate is a polished silicon wafer, alumina, or glass or a material coated with one or more of these materials. For example, a substrate may be a polycarbonate membrane coated with a layer of amorphous silicon, a fibrous material coated with aluminum or chromium and an optical layer of amorphous silicon, or a ceramic coated with a layer of metal and/or amorphous silicon. The primary consideration for selection of the substrate is the reflectivity of the material and/or its ability to be coated with a reflective material.
The term xe2x80x9coptical pathwayxe2x80x9d as used herein refers to a pathway within a device through which electromagnetic radiation may pass. The optical pathway serves to direct electromagnetic radiation from a light source to a sample under study, and ultimately to a detector that measures one or more properties (e.g. intensity, polarization, etc.) of light that is reflected by the sample. The optical pathway may contain various elements of the device, such as polarization elements that are positioned to polarize incoming electromagnetic radiation from the light source prior to contact with the sample under study, and/or electromagnetic radiation reflected from the sample under study. The optical pathway preferably includes only those components that are required to permit the detector to provide signals that facilitate qualitative measurements of film thickness. Such devices can be cost-effectively deployed for applications where ellipsometers have not been traditionally used, for example, in the physician""s office.
The term xe2x80x9clight sourcexe2x80x9d as used herein refers to any source of electromagnetic radiation. Electromagnetic radiation can also be referred to as xe2x80x9clight.xe2x80x9d Such electromagnetic radiation may include wavelengths from about 10xe2x88x926 xcexcm to about 108 xcexcm, preferred is electromagnetic radiation from the ultraviolet to infrared wavelengths; particularly preferred electromagnetic radiation is visible light. Suitable light sources are well known to those skilled in the art, and can include any source of monochromatic or polychromatic radiation. The use of monochromatic radiation is preferred. The terms xe2x80x9cmonochromatic radiationxe2x80x9d or xe2x80x9cmonochromaticxe2x80x9d light as used herein refer to electromagnetic radiation having a bandwidth that is sufficiently narrow to function as a single wavelength for design purposes. Preferred light sources are lasers, laser diodes, and light emitting diodes.
As used herein, the term xe2x80x9cdetectorxe2x80x9d refers to any device for detecting electromagnetic radiation by the production of electrical or optical signals, and includes photomultipliers, photodiodes, and photochemical reagents, whether these detectors are driven to provide analog or digital signals, as well as any other light detection device. Preferred detectors detect electromagnetic radiation, particularly visible light, with the resulting production of electrical or optical signals. A signal processing element can process these signals to yield this information, for example by the use of standard curves, to associate the signals with a film thickness. In especially preferred embodiments, the film thickness is interpreted as a binding assay result, e.g., the result of a test showing either a positive, negative, or inconclusive result in a test for a specific analyte.
The term xe2x80x9cpolarization elementxe2x80x9d as used herein refers to a device that receives incoming electromagnetic radiation, and produces therefrom radiation which is polarized. Suitable polarization elements, such as polarizing filters and analyzers, are well known to those skilled in the art. As described herein, polarization elements can be positioned to polarize incoming light from the light source prior to contact with the sample under study, as well as light reflected from the sample under study. A polarization element can be fixed within the optical pathway. Alternatively, one or more of the polarization elements can include a mechanism for varying s- and p-components of polarized light with time by rotating the polarization element, or a component thereof, on its optical axis. Preferably, this mechanism rotates a polarizing filter that is located in the position of a polarizer or analyzer in a conventional ellipsometer. Rotation of a polarizing filter provides a corresponding quasi-sinusoidal intensity in the electromagnetic radiation that is reflected from the sample under study.
The term xe2x80x9clinear polarizationxe2x80x9d as used herein refers to a polarization state that is essentially all s-polarization or all p-polarization. Electromagnetic radiation is linearly polarized if, in either linear state, there is not enough of the other polarization state to affect the outcome of the measurement. Preferably, a linear polarizing filter may be rotated up to about 20xc2x0 rotation off of its optical axis without introducing appreciable measurement errors; more preferably, this rotation is limited to less than about 10xc2x0; even more preferably, limited this rotation is limited to less than about 5xc2x0, with a precise alignment of about 1xc2x0 or less being most preferred.
In another aspect, the invention concerns methods of measuring a film thickness of a sample. The methods can comprise providing a device comprising a light source, a polarizer, an analyzer, and a detector; directing electromagnetic radiation from the light source towards the sample, whereby electromagnetic radiation is reflected from the sample; polarizing the electromagnetic radiation that is directed towards the sample using the polarizer; polarizing the electromagnetic radiation reflected from the sample using the analyzer; rotating the polarizer or the analyzer to vary the s and p content of the polarized electromagnetic radiation with time; detecting the polarized electromagnetic radiation reflected from the sample using the detector to obtain a signal corresponding to the intensity of the reflected electromagnetic radiation; and correlating the signal to the film thickness of the sample using a standard function that relates film thickness to detector signal intensity. Other data analysis means can be used as known to those skilled in the art.
In particularly preferred embodiments, one or more of the following may be included in the methods: (i) a standard function selected from a plurality of standard functions obtained from samples having different optical properties, (ii) a comparative detector signal intensity obtained from a negative control sample, (iii) a standard function comprising a normalizing function which relates the detector signal intensity to the comparative detector signal intensity, (iv) a normalizing function that is a ratio of the detector signal intensity and the comparative detector signal intensity, and (v) a polarizer or an analyzer that provides a corresponding quasi-sinusoidal signal from the detector.
Selected signals can be obtained in a time domain corresponding to predetermined degrees of rotation of the polarizing filter, or other polarization element, which varies the s and p content of the polarized light. These intensity signals are used as input to generate a standard function, e.g., standard curves of empirical or theoretical data that relate film thickness as a function of the magnitude of detector signal intensity through a range of polarizing rotation. Any mapping technique may be used to relate or map the intensity corresponding to a particular degree of rotation with a film thickness. These other mapping techniques may include, without limitation, neural networks and adaptive filters, which are all referred to in the context of this application as xe2x80x9cstandard functionsxe2x80x9d.
A particular advantage that derives from the ellipsometric embodiments of the invention is that operation of the device does not require time consuming positional adjustment of the polarization element in order to optimize intensity in signals from the detector. More particularly, measurement data can be collected from all points in the cycle of polarizing rotation and used to establish a peak to peak amplitude of the quasi-sinusoidal signal. This peak to peak amplitude is used as input into the standard curve in order to determine film thickness.
A particularly preferred feature of the ellipsometric embodiments is the use of a normalizing function that expresses the detector signal intensity in relationship to a comparative detector signal intensity obtained from a negative control sample. This normalizing function is most preferably a ratio between the detector signal intensity and the comparative signal intensity from the negative control sample, e.g., the detector signal intensity divided by the intensity of the signal from the negative control sample.
In another aspect, the invention concerns devices for use in interpreting thin film binding assays which comprise a substrate for supporting a sample; a light source for producing electromagnetic radiation to illuminate the sample; a detector for detecting electromagnetic radiation reflected from the sample; an optical pathway between the light source, the sample, and the detector; and a signal processor for correlating the signal with a film thickness on the sample. The optical pathway comprises a fixed polarization element located between the sample and the detector to linearly polarize the reflected electromagnetic radiation. A signal produced by the detector corresponds to the intensity of the electromagnetic radiation reflected from the sample.
In particularly preferred embodiments, one or more of the following can be include d in the device: (i) a light source that produces monochromatic electromagnetic radiation, (ii) electromagnetic radiation preferably selected from the group comprising visible light, infrared light, and ultraviolet light, (iii) linearly polarized electromagnetic radiation that is essentially s- or p-polarized relative to a plane of incidence in the optical pathway between the polarizer and the detector, (iv) relating a film thickness to a thin film binding assay result, and (v) an optical pathway comprising a single polarization element.
In another aspect, the invention concerns methods of measuring a film thickness of a sample which comprise providing a device comprising a light source, a detector, a first optical pathway between the light source and a sample, and a second optical pathway between the sample and the detector; directing electromagnetic radiation from the light source along the first optical pathway to the sample, such that electromagnetic radiation is reflected by the sample along the second optical pathway to the detector; linearly polarizing the electromagnetic radiation along the first optical pathway at a position prior to contact of the electromagnetic radiation with the sample; detecting the electromagnetic radiation reflected by the sample using the detector to obtain a signal corresponding to the intensity of the reflected electromagnetic radiation; and correlating the signal to the film thickness of the sample.
In particularly preferred embodiments, one or more of the following can be included in the methods: (i) linearly polarized electromagnetic radiation that is essentially s- or p-polarized relative to a plane of incidence in the second optical pathway between the polarizer and the detector, (ii) relating a film thickness to a thin film binding assay result, and (iii) performing the method without polarization of the electromagnetic radiation reflected from the sample.
In another aspect, the invention concerns methods of measuring a film thickness of a sample which comprises providing a device comprising a light source, a detector, a first optical pathway between the light source and a sample, and a second optical pathway between the sample and the detector; directing electromagnetic radiation from the light source along the first optical pathway to the sample such that electromagnetic radiation is reflected by the sample along the second optical pathway to the detector; linearly polarizing the electromagnetic radiation along the second optical pathway at a position after contact of the electromagnetic radiation the said sample; detecting the electromagnetic radiation reflected by the sample using the detector to obtain a signal corresponding to the intensity of the reflected electromagnetic radiation; and correlating the signal to the film thickness of the sample.
In particularly preferred embodiments, one or more of the following can be included in the methods: (i) linearly polarized electromagnetic radiation that is essentially s-or p-polarized relative to a plane of incidence in the second optical pathway between the polarizer and the detector, (ii) relating a film thickness to a thin film binding assay result, and (iii) performing the method without polarization of the electromagnetic radiation prior to reflection from the sample.
An optical pathway according to the invention includes specific combinations of elements that operate according to the principles of multiple thin-film reflection theory. In a preferred embodiment, the optical pathway comprises a polarizing filter that is positioned to linearly polarize light prior to its illumination of the sample under study. In other preferred embodiments, the optical pathway comprises a polarizing filter that is positioned to linearly polarize light that is reflected from the sample under study.
In these two instrument configurations, the polarization element, either in the path of the incident electromagnetic radiation or in the path of reflected electromagnetic radiation, is used to select one component of the polarized electromagnetic radiation, i.e., either the s- or the p-component. When the polarizing element is in the path of the incident electromagnetic radiation, the s- or p-polarized electromagnetic radiation is incident on the sample under study. Upon interaction with the sample under study, the electromagnetic radiation undergoes an amplitude change relative to electromagnetic radiation that has reflected from a sample under study without the added thin film, e.g. relative to a negative control sample. The reflected electromagnetic radiation is not elliptically polarized, and there is no change in the degree of polarization. Rather, there is only an attenuation of the light relative to the light that is reflected from a sample that does not have an added thin film. A similar case is generated when the incident electromagnetic radiation, and thus the reflected electromagnetic radiation, is unpolarized. In such a case, a polarization element in the path of the reflected electromagnetic radiation will pass only one component of the electromagnetic radiation to the detector.
In another aspect, the invention concerns devices for use in interpreting thin film binding assays which comprises a substrate for supporting a sample, a light source for producing electromagnetic radiation to illuminate the sample, a detector for detecting electromagnetic radiation reflected from the sample, an optical pathway between the light source, the sample, and the detector, and a signal processor for correlating said signal with a film thickness on said sample. A signal produced by the detector corresponds to the intensity of electromagnetic radiation reflected from the sample.
In particularly preferred embodiments, one or more of the following can be included in the devices: (i) a light source that produces monochromatic electromagnetic radiation, (ii) electromagnetic radiation selected from the group comprising visible light, infrared light, and ultraviolet light, (iii) a light source positioned relative to the sample and the detector to provide a low angle of incidence, (iv) an optical pathway comprising neither a polarizer located between the light source and the sample, nor a polarizer located between the sample and the detector, (v) a light source positioned at an angle of incidence ranging from about 0xc2x0 to about 30xc2x0, determined relative to a line normal to the plane of the sample, (vi) a light source positioned at an angle of incidence ranging from 0xc2x0 to 20xc2x0, (vii) a light source positioned at an angle of incidence ranging from 0xc2x0 to 10xc2x0, (viii) relating a film thickness to a thin film binding assay result, (ix) an optical pathway comprising a polarizing filter which provides circularly polarized light, (x) a polarizing filter located in the optical pathway between the light source and the sample, and (xi) a polarizing filter located in the optical pathway between the sample and the detector.
In another aspect, the invention describes a method of measuring a film thickness of a sample which comprises providing a device comprising a light source, a detector, a first optical pathway between the light source and a sample, and a second optical pathway between the sample and the detector; directing electromagnetic radiation from the light source along the first optical pathway to the sample such that electromagnetic radiation is reflected by the sample along the second optical pathway to the detector; detecting the electromagnetic radiation reflected by the sample using the detector to obtain a signal corresponding to the intensity of the reflected electromagnetic radiation; and correlating the signal to the film thickness of the sample. Preferably, the electromagnetic radiation is unpolarized at said detector without movement of components in said optical pathway.
In particularly preferred embodiments, one or more of the following can be included in the method: (i) a light source positioned at a low angle of incidence determined relative to a line normal to the sample, (ii) an optical pathway comprising neither a polarizer located between the light source and the sample, nor a polarizer located between the sample and the detector, (iii) a low angle of incidence ranging from about 0xc2x0 to about 30xc2x0, (iv) a low angle of incidence ranging from 0xc2x0 to 20xc2x0, (v) a low angle of incidence ranging from 0xc2x0 to 10xc2x0, and (vi) relating a film thickness to a thin film binding assay result.
In these preferred embodiments, the invention describes devices in which the light source and the detector are positioned in the device at angles of incidence where essentially unpolarized reflected light to the detector can be used. In this device configuration, the light source can be positioned at an angle of incidence ranging from about 0xc2x0 to about 30xc2x0, determined relative to a line normal to the plane of the sample. Preferably, the angle of incidence can be about 0xc2x0, about 5xc2x0, about 10xc2x0, about 15xc2x0, about 20xc2x0, about 25xc2x0, or about 30xc2x0. The wavelength of the electromagnetic radiation to be provided can be determined empirically, using multilayer thin film reflection theory that is well known in the art. Using such theory, the selected wavelength is a function of the angle of incidence, the approximate thickness of the thin film on the sample under study, and the reflective surface that is used to support the thin film.
In any of the reflectometric embodiments, it is preferred to have no additional polarizing means other than those that are specifically mentioned. For example, in the first reflectometric embodiment where light is polarized prior to contact with the sample under study, it is preferred not to have a polarizing filter located between the sample and the detector. Similarly, in the second reflectometric embodiment where light is polarized after contact with the sample under study, it is preferred not to have a polarizing filter located between the sample and the light source. In the third reflectometric embodiment, it is preferred not to have any polarizing filters.
The most cost effective devices described in the invention simply mount a light source on a housing or frame that positions the light source relative to the substrate and the detector in a manner providing a suitable angle of incidence. Reflected light from the substrate arrives at the detector relative to the incident light or light that is reflected from the surface without a thin film. In this case, the light source is a monochromatic light source and the combination of elements includes neither a polarizer located between the light source and the sample under study nor an analyzer located between the sample under study and the detector.
A particular advantage of the present invention is that operation of the reflectometric embodiments do not require positional adjustment of the polarization elements. Removing one or more of the polarizing elements from the instrument""s optical path increases the amount of light available to the detector. Thus, the instrument is more sensitive than conventional instruments, where light is lost by passage through the polarizing elements. More particularly, measurement data is collected as a detector signal intensity that is compared to a delimiting value. This delimiting value is associated with a background or negative test result or other indicator of film thickness change. In its most fundamental form, the interpretation of test results is essentially an answer of yes or no, positive or negative, based upon signal intensity measurements showing the presence or absence of a thin film or the presence of a film with a thickness greater than a preselected threshold thickness. In such a preferred embodiment, actual film thickness does not need to be calculated, provided the test results conclusively establish the presence or absence of such a film. Because the signal is a function of the film thickness, and the film thickness is a function of analyte concentration in a sample, the instrument can also provide a quantitative determination of analyte concentration without an absolute determination of the film thickness.
It is also contemplated that the test interpretation could include a third indicator, namely, an indicator that the test is inconclusive because the signal intensity falls within a range of values between a definite negative and a definite positive.
In addition to assays where direct detection of analyte binding results in an increase in thickness on the thin film support, assays where a decrease in thickness or where the analyte concentration is inversely related to signal can be envisioned. Examples of such assays would include enzymatic degradation of the thin film surface where the substrate is specific to an enzyme as the analyte of interest or a competitive assay where the analyte competes with the amplifying reagent and the thickness change decreases with increasing analyte concentration.
Also instrument response to a change in thickness can be an increase in light intensity to the detector or a decrease in light intensity to the detector.